Sample Size Considerations in Clinical Trials When Comparing Two Interventions Using Multiple Co-Primary Binary Relative Risk Contrasts

<div><p>The effects of interventions are multidimensional. Use of more than one primary endpoint offers an attractive design feature in clinical trials as they capture more complete characterization of the effects of an intervention and provide more informative intervention comparisons. For these reasons, multiple primary endpoints have become a common design feature in many disease areas such as oncology, infectious disease, and cardiovascular disease. More specifically in medical product development, multiple endpoints are used as co-primary to evaluate the effect of the new interventions. Although methodologies to address continuous co-primary endpoints are well-developed, methodologies for binary endpoints are limited. In this article, we describe power and sample size determination for clinical trials with multiple correlated binary endpoints, when relative risks are evaluated as co-primary. We consider a scenario where the objective is to evaluate evidence for superiority of a test intervention compared with a control intervention, for all of the relative risks. We discuss the normal approximation methods for power and sample size calculations and evaluate how the required sample size, power, and Type I error vary as a function of the correlations among the endpoints. Also we discuss a simple, but conservative procedure for appropriate sample size calculation. We then extend the methods allowing for interim monitoring using group-sequential methods. Supplementary materials for this article are available online.</p></div

¹¹C-Acetate PET Imaging in Patients with Multiple Sclerosis

<div><p>Background</p><p>Activation of glial cells is a cardinal feature in multiple sclerosis (MS) pathology, and acetate has been reported to be selectively uptaken by astrocytes in the CNS. The aim of this study was to investigate the efficacy of PET with ¹¹C-acetate for MS diagnosis.</p><p>Materials and Methods</p><p>Six patients with relapsing-remitting MS and 6 healthy volunteers (HV) were enrolled. The ¹¹C-acetate brain uptake on PET was measured in patients with MS and HV. Volume-of-interest analysis of cerebral gray and white matter based on the segmentation technique for co-registered MRI and voxel-based statistical parametric analysis were performed. Correlation between ¹¹C-acetate uptake and the lesion number in T1- and T2- weighted MR images were also assessed.</p><p>Results</p><p>The standardized uptake value (SUV) of ¹¹C-acetate was increased in both white and gray matter in MS patients compared to HV. Voxel-based statistical analysis revealed a significantly increased SUV relative to that in the bilateral thalami (SUVt) in a broad area of white matter, particularly in the subcortical white matter of MS patients. The numbers of T2 lesions and T1 black holes were significantly correlated with SUV of ¹¹C-acetate in white and gray matter.</p><p>Conclusions</p><p>The ¹¹C-acetate uptake significantly increased in MS patients and correlated to the number of MRI lesions. These preliminary data suggest that ¹¹C-acetate PET can be a useful clinical examination for MS patients.</p></div

Correlation between ¹¹C-acetate SUV and the number of MRI lesions in patients with MS.

<p>Correlation between ¹¹C-acetate SUV in WM or GM and the number of T1 black holes (A, C) or T2 lesions (B, D) in each hemisphere of the six MS patients. SUV: standardized uptake value.</p

¹¹C-acetate CNS biodistribution.

<p>(A)Mean standardized uptake value (SUV) of each lesion. (B) Relative SUV compared to that of the thalamus (SUVt). Data are expressed as the mean ± standard error of the mean (SEM) (n = 6). The Mann–Whitney <i>U</i> test showed a significant difference in the median between the HV and MS groups (*:p<0.0055 after Bonferroni correction). HV  =  healthy volunteers, MS  =  multiple sclerosis.</p

¹¹C-acetate uptake distribution and quantification in MS patients.

<p>(A)Spatially normalized group mean images of ¹¹C-acetate SUVt automatically segmented based on MRI. VOI analysis summarizing the mean SUVt in WM (B) and GM (C), and the WM/GM SUV ratio (D) in the HV and MS groups. The identical analysis performed using spill-in-free VOIs are also shown (E–G). The p-value was calculated using the analysis of covariance to adjust the variance of age. (H) The SPM analysis result is overlaid onto the T1-weighted brain MRI template. Colored voxels indicate T-scores representing significantly increased ¹¹C-acetate uptake (SUVt) in patients with MS compared to HV patients. The spatially normalized PET images were smoothed for the analysis using a 12-mm FWHM isotropic Gaussian kernel. The significance thresholds are corrected for multiple comparisons at the cluster level with a p-value of 0.05 (family-wise error correction). SUV: standardized uptake value.</p

Administration of R037 inhibits the secretion of inflammatory cytokines.

<p>Lymphocytes were isolated by draining lymph nodes (dLN) and spleens of C57BL/6 mice on day 11 after immunization and restimulating with MOG_35–55 for 72 h. IL-17, IFN-γ and IL-10 in the culture supernatants were assayed by ELISA. Decreased IL-17 production by both dLN cells and splenocytes and decreased IFN-γ by splenocytes were observed in the R037-treated group. Data are shown as mean + SEM from a representative of three independent experiments for 6 mice in the R037-treated and 8 in the control groups. **p≤0.01; *p≤0.05.</p

Administration of R037 induces CD4⁺ IL-10-producing T cells in mesenteric lymph nodes (MLNs) and spleen.

<p>(A) Lamina propria cells, MLNs and splenocytes were isolated from the unimmunized mice that were fed R037 for 2 weeks and after isolation, cells were stimulated with anti-CD3/anti-CD28 antibodies for 72 h in vitro. IL-17, interferon-γ (IFN-γ) and IL-10 in the supernatants were assayed by ELISA. For lamina propria, each bar indicates mean + SEM of triplicate samples from 2 mice in each group. For MLNs and splenocytes, each bar indicates mean + SEM of 8 mice in each group. Data are representative of more than three independent experiments for 8 mice each. * p≤0.05 (B) Lamina propria cells, MLNs and splenocytes were isolated from the mice that were fed R037 for 2 weeks. Intracellular staining of IL-10 and Foxp3 (B) in CD4⁺ T cells was analyzed by flow cytometry. Data are representative of 2 independent experiments.</p

<i>Pediococcus acidilactici</i> ameliorates EAE.

<p>(A) R037 (4 mg/day; n = 18) or PBS (n = 17; control) was administered daily by oral gavages to C57BL/6 mice from 14 days before immunization with MOG_35–55 until the end of the study. (B) R037 (0.8 mg/ml; n = 16) or PBS (n = 17) in a water bottle was administered to SJL/J mice from 14 days before immunization with PLP_131–151 until the end of the study. Data represent mean score + SEM from a representative of two independent experiments. *p≤0.05; **p≤0.01 (t-test).</p

Infiltration of MNCs into the spinal cord is reduced in R037-treated mice.

<p>Spinal cord sections obtained from control (A) or R037-treated (B) C57BL/6 mice on day 22 after immunization were analyzed by hematoxyline and eosin (H&E) staining. Scale bar = 250 µm. (C) Semiquantitative evaluation of the pathological scores was performed as described in the Methods section. Each bar indicates the mean pathological score + SEM of 8 mice from each group.</p

R037 relieves EAE severity in a therapeutic manner.

<p>R037 (0.8 mg/ml; n = 16) or PBS (n = 17; control) were administered in a water bottle to C57BL/6 mice from 11 days after immunization with MOG_35–55. The area under the curve (AUC) under the bar was significantly lower in R037-treated mice. Data represent mean score + SEM of two independent experiments. *p≤0.05.</p